1. Field of the Art
The present invention relates to a DNA sequence coding for a so-called signal peptide and to its use for producing proteins by means of genetic engineering. More particularly, the present invention relates to a process for producing proteins with us of Bacillus brevis as a host and also to a DNA sequence required therefor.
2. Related Art
A method for producing heterologous proteins in microorganisms with use of recombinant DNA techniques has been widely used for the production of medical and pharmaceutical products.
It is Escherichia coli that has been most popularly used as a host microorganism for such a method. However, the heterologous protein produced is usually retained within cells of E. coli as the host. Thus, it is required for the purification of the heterologous protein to destroy the cells and to remove a number of compounds such as proteins derived from E. coli or the like. Furthermore, the heterologous proteins which are produced in abundance in the limited space of the cells sometimes form inclusion bodies, so that great efforts are often required to regenerate activities of the heterologous proteins.
On the other hand, in a system in which Bacillus subtilis, yeasts or the like are used as host microorganisms, there is an advantage that heterologous proteins produced are generally secreted extracellularly from the host cells and thus the products are easily recovered. Such an advantage in Bacillus subtilis or the like as the host presumably depends on a mechanism that the host microorganism has a gene carrying specific genetic informations and produces a desired protein as a precursor in which peptide chains, so-called signal peptides, are bound, and the precursor is passed through the cell membrane and secreted extracellularly or into the periplasm, whereupon the signal peptide chains are cleaved off and mature proteins are obtained.
As an example of a system in which a microorganism of genus Bacillus is used, there has bee reported a successful example of the secretion of a large amount of foreign genetic products into a culture medium by using Bacillus brevis 47 which secretes a protein in an amount as much as 12 g/l into the medium under the optimal culture condition [S. Udaka, (1976) Agric. Biol. Chem., 40, 523-528; S. Miyashiro, H. Enei, K. Tekeinami, Y. Hirose, T. Tsuchida and S. Udaka (1980), Agric. Biol. Chem., 44, 2297-2303], linking a promotor of a gene coding for MWP which is one of the proteins secreted by the microorganism and a DNA coding for a signal peptide with a foreign gene such as human epidermal growth factor or the like and introducing the linked DNA into the Bacillus brevis 47 [H. Yamagata, et al., (1989), Proc. Natl. Acad. Sci. U.S.A., 86, 3589-3593].
However, the foreign genetic products thus produced may possibly be decomposed in a culture medium because of the proteolytic enzymes extracellularly secreted in a small amount by the Bacillus brevis 47.